Comparison of Fluorescence in Situ Hybridization and Primed in Situ Labeling Methods for Detection of Single-Copy Genes in the Fungus Ustilago maydis

Abstract

Li, S., Harris, C. P., and Leong, S. A. 1993. Comparison of fluorescence in situ hybridization and primed in situ labeling methods for detection of single-copy genes in the fungus Ustilago maydis. Experimental Mycology 17, 301-308. This report describes the use of fluorescence in situ hybridization for detection of single-copy genes in the fungus, Ustilago maydis . Either biotin- or digoxigenin-labeled DNA probes were hybridized to target nuclei of sporidia and subsequently rendered fluorescent by successive treatments with fluorescein (FlTC)-labeled avidin and biotinylated anti-avidin antibody or with anti-digoxigenin-FlTC or anti-digoxigenin-rhodamine. These results showed that the digoxigenin-based hybridization can be used successfully to specifically detect single-copy genes in nuclei of U. maydis with either 3.6- or 6.7-kb genomic DNA probes. By contrast, the biotin-based hybridization technique gave rise to nonspecific background. Primed in situ labeling with fluorescein-12-dUTP was also used successfully to specifically detect a 1.5-kb fragment of single-copy genomic DNA in nuclei of U. maydis sporidia. This technique has the advantages of lower staining background, shorter time of analysis, and a higher efficiency of hybridization of shorter DNA probes to target DNA, when compared to traditional in situ hybridization.